Project 3, Abstract Transcript processing and degradation are key processes by which the cell regulates its functional capacity and physiological state, yet little is known about how these pathways function in Mycobacterium tuberculosis (Mtb). Mutations in components of the RNA processing and degradation machinery are associated with drug resistance in clinical strains and with drug tolerance in mice and in vitro, highlighting the relevance of these pathways for clinically important phenotypes. The lack of knowledge about the fundamental biology of mycobacterial RNA metabolism, including pathway structure and functional consequences of these pathways, represent a barrier to understanding clinically important routes to drug resistance. The proposed project addresses this gap by elucidating the targets and functional relationships between the critical RNA processing and degradation proteins, focusing on how these processes modify drug efficacy. The specific aims are to: 1. Determine the physical and genetic interactions that define and organize RNA processing and degradation pathways. 2. Define the targets of RNA degradation proteins that are associated with drug resistance. 3. Determine the phenotypic consequences of perturbations to RNA processing pathways. The project seeks to move from piecemeal efforts to a pathways-directed approach capable of elucidating the consequences of complex processes important to adaptation to the host and to drug pressure. To achieve this, the project leverages high-throughput methodologies and extensive collaboration with other projects and cores. Biochemical approaches will be used to define the physical associations between degradation pathway components and their target specificities; TnSeq to map pathway structure and its phenotypic consequences; transcriptomics approaches to determine how RNA metabolism pathways shape the transcriptome; murine models to interrogate the relationship between RNA processing mutants and drug efficacy in vivo; and metabo- lipidomics approaches to determine the impact of RNA processing mutants on metabolic status and permeability. Combining a variety of approaches and expertise in an intentional and systematic fashion will allow the project to define the mechanisms and consequences of RNA processing and degradation in mycobacteria in a way that has not previously been feasible. In the long term, the knowledge obtained will inform efforts to design more effective diagnostics, drugs and regimens.